Midazolam Oxidation by Cytochrome P450 3A4 and Active-Site Mutants: an Evaluation of Multiple Binding Sites and of the Metabolic Pathway That Leads to Enzyme Inactivation
Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding...
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Published in | Molecular pharmacology Vol. 61; no. 3; pp. 495 - 506 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Pharmacology and Experimental Therapeutics
01.03.2002
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Subjects | |
Online Access | Get full text |
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Summary: | Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual
residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that
oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K I and k inact values of 5.8 μM and 0.15 min â1 , respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1â²-OH MDZ as opposed
to 4-OH MDZ provided strong evidence that the 1â²-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1â²-OH
formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of
MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino
acid caused an increase in the ratio of 1â²-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210,
Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants
revealed that the alteration in regioselectivity is caused by a change in kinetic parameters ( V max and K M ) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the
regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication
that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.61.3.495 |